Phosphoserine/threonine-binding domains and protein kinases play critical roles in controlling multiple aspects of cell proliferation, including cell cycle progression and the cellular response to DNA damage. The long-term goal of our laboratory is to identify and characterize these phosphopeptide domains and protein kinases with a focus on elucidating the molecular basis for their functions in cell cycle control. One family of protein kinases, Polo-like kinases, play critical roles during multiple stages of the eukaryotic cell cycle, including many of the events that occur during M-phase, as well as in the DNA damage response. Polo-like kinases are up-regulated in many types of human cancer, and their experimental down-regulation results in decreased cell proliferation and tumor regression, making them ideal targets for anti-cancer drug development. Our laboratory recently discovered that the C-terminal domain of Polo-like kinases, which we have named the Polo-box domain, is a phosphoserine/threonine-binding domain that is critical for targeting Polo-like kinases to their substrates. We have determined the optimal phosphopeptide sequence motif recognized by the Polo-box domain, and solved the high-resolution X-ray crystal structure of the Polo-box domain in complex with its optimal phosphopeptide. In this grant application we propose to develop a high throughput assay suitable for identifying small molecules that interfere with substrate targeting by the Polo box domain, or substrate phosphorylation by the kinase domain. The development of these assays should allow rapid screening of small molecule Polo kinase inhibitors that will function as novel anti-cancer agents. [unreadable] [unreadable]